A great number of analytic instruments based upon the optical properties of a liquid sample have been proposed to the arts. In common such instruments provide an optically transparent path for the beam from an emitter of the radiation to a receptor. The liquid sample to be analyzed is interposed across the beam so that the radiation received by the receptor has been altered (in some optical sense) by interposition of an optical path through the liquid sample. Typically the optical system employs infrared, visible or ultraviolet radiation and measures optic changes caused by passage through the liquid, e.g. the degree to which monochromatic ultraviolet light has been absorbed by the sample. One notable and widely used optical system measures UV absorption of the 254 nm mercury line, by the sample. Another notable optical system provides detection in the infrared region of the spectra of the sample. Still other optical systems employ polarimetry, refractive index, turbidimetry (nephelometry) or fluorimetry for analytic purposes.
The present invention can be employed in the optical path of any optical technique instrument wherein the least length optical path through a liquid is desirable. For each of discussion, this invention will be described in detail in terms of use within the context of liquid chromatography with a UV detector system (a preferred embodiment).
As is well known to the art (see "Introduction of Modern Liquid Chromatography" by R. L. Snyder and J. J. Kirkland; John Wiley & Sons, 1974) liquid chromatography involves chromatographic separation of a test sample in a liquid moving phase by adsorption, partition or ion exchange, typically in a column. The chromatographed solution is then analyzed by measuring absorption of ultraviolet light in the chromatographed liquid as the UV traverses the optical path therethrough.
The laws of Absorption in Spectroscopy state that the fractional part of the monochromatic radiant energy, or intensity, absorbed in a thin layer of material depend upon the substance and upon the frequency of the incident radiation and is proportional to the thickness, i.e. Beer's Law: EQU A=.epsilon.bc
where A=absorbance, .epsilon.=molar absorptivity, b=cell (or path) length in cm, and c=concentration in moles/L. Further, absorbance is defined relative to the incident radiant energy, P.sub.o and the transmitted energy, P, as: ##EQU1##
Materials which have a large absorbance value, e.g. long conjugated molecules, particularly in high concentration, are difficult to analyze with standard spectrophotometers due to the large extinction coefficients (molar absorptivity), i.e. the point at which the light passing through the sample is effectively extinguished.
In liquid chromatographic detection, as in normal absorption spectroscopy, the common optical path length is 10 mm. In preparative LC work, the samples get rather large and thus the concentration of highly absorptive substance in the detector are high. The result is excessively large absorbance values. If, for example, the molar absorptivity, .epsilon., of an LC sample is 3,000 (a typical, high value), and if the concentration in the detector attains a maximum value of 7.times.10.sup.-3 M, and a 1.0 cm cell is used, the absorbance calculates out to: ##STR1##
Such a value, (21), is much too high for most spectrophotometers. Detectors in LC systems respond to changes in concentration of the sample substance in the solvent. Within instrument limits such response is linear, i.e. straight line. Suffice is here to merely point out that the linear response is proportional to the size of the various samples intended for the LC system, and the linear response is plotted as a straight line. However, at some concentration of the sample substance in the solvent all detectors respond in a non-linear manner. A detector is considered overloaded when additional quantities of (a pure) sample no longer result in an increased response or signal. Since the typical LC system exhibits a linear response only up to about 2.0 A, the value of 21 calculated out above is very much of an overload.
Analytic instruments employing an optical characteristic for measurement purposes have included heretofore a closed chamber or cell formed from materials transparent to the radiation employed (e.g. glass, quartz) positioned across the beam of radiation inside the instrument. The chromatographed solution is, of course, passed through the cell, and the cell dimensions are what fix the optical path length, (e.g. thickness "b" for absorptivity measurement). The usual cell length is 2-10 mm. The nature of liquid flow through capillary sized channels preclude substantial reduction in cell dimensions e.g. to below about 1 mm. Moreover, accurately dimensioned short optical path cells of below about 1 mm would be quite expensive to fabricate.
None the less an optical path (length "b") of very small dimensions would be advantageous for optical measurements on materials with very large molar absorptivity values and/or high concentration levels. If the path length is reduced to 0.1 cm (1 mm) the absorbance calculated above decreases to 2.1, a value reasonable for most spectrophotometers. A further decrease in path length to 0.01 cm (0.1 mm) would reduce the absorbance to 0.21, which is a very low value.
Thus, by a substantial reduction in the length of the optical path, the A (Absorbance) is lowered significantly. High absorbancy samples cain be held to the range of linearity of existing UV detectors (about 0.001 A to 2.0 A). an optical path of about 0.1 mm, a path length desired for analyzing samples with high absorbance, may be attained by practice of this invention.